Neoplasia is a process that occurs in cancer, by which the normal controlling mechanisms that regulate cell growth and differentiation are impaired, resulting in progressive growth. This impairment of control mechanisms allows a tumor to enlarge and occupy spaces in vital areas of the body. If the tumor invades surrounding tissue and is transported to distant sites (metastases) it will likely result in death of the individual.
The desired goal of cancer therapy is to eliminate cancer cells preferentially, without having a deleterious effect on normal cells. Several methods have been used in an attempt to reach this goal, including surgery, radiation therapy and chemotherapy.
Local treatments, such as radiation therapy and surgery, offer a means of reducing the tumor mass in regions of the body that is accessible through surgical techniques or high doses of radiation therapy. However, more effective local therapies with fewer side effects are needed. Moreover, these treatments are not applicable to the destruction of widely disseminated or circulating tumor cells eventually found in most cancer patients. To combat the spread of tumor cells, systemic therapies are used.
One such systemic treatment is chemotherapy. Chemotherapy is the main treatment for disseminated, malignant cancers. However, chemotherapeutic agents are limited in their effectiveness for treating many cancer types, including many common solid tumors. This limitation is in part due to the intrinsic or acquired drug resistance of many tumor cells. Another drawback to the use of chemotherapeutic agents is their severe side effects. These include bone marrow suppression, nausea, vomiting, hair loss, and ulcerations in the mouth. Clearly, new approaches are needed to enhance the efficiency with which a chemotherapeutic agent can kill malignant tumor cells, while at the same time avoiding systemic toxicity.
H19 in Diagnosis and Therapy
The H19 gene is one of several genes known to be imprinted in humans (Hurst et al., 1996, Nature Genetics 12:234 237). At the very beginning of embryogenesis, H19 is expressed from both chromosomal alleles (DeGroot et al., 1994, Trophoblast 8:285 302). Shortly afterwards, silencing of the paternal allele occurs, and only the maternally inherited allele is transcribed.
H19 is abundantly expressed during embryogenesis, and was first identified as a gene that was coordinately regulated with alpha-fetoprotein in liver by the trans-acting locus raf (Pachnis et al., 1984, “Locus unlinked to alpha-fetoprotein under the control of the murine raf and Rif genes”, Proc Natl Acad Sci. 81:5523 5527). Additionally, H19 has been independently cloned by several groups using screens aimed at isolating genes expressed during tissue differentiation. For example, the mouse homolog of H19 was identified in a screen for genes that are active early during differentiation of C3H10T1/2 cells (Davis et al., 1987, “Expression of a single transfected cDNA converts fibroblasts to myoblasts”, Cell 51:987 1000). Similarly, murine H19 was shown to be expressed during stem cell differentiation and at the time of implantation (Poirier et al., 1991, “The murine H19 gene is activated during embryonic stem cell differentiation in vitro and at the time of implantation in the developing embryo”, Development 113:1105 1114). Transcription of the human H19 gene was also discovered in differentiating cytotrophoblasts from human placenta (Rachmilewitz et al., 1992, Molec. Reprod. Dev. 32:196 202).
While transcription of H19 RNA occurs in many different embryonic tissues throughout fetal life and placental development, H19 expression is downregulated postnatally, although low levels of H19 transcription have been reported, for example, in murine adult muscle and liver (Brunkow and Tilghman, 1991, “Ectopic expression of the H19 gene in mice causes prenatal lethality”, Genes Dev. 5:1092 1101).
H19 transcription can be re-activated postnatally in cancer cells as demonstrated in tumors derived from tissues expressing H19 prenatally (Ariel et al., 1997, “The product of the imprinted H19 gene is an oncofetal RNA”, Mol Pathol. 50:34 44). Additionally, H19 RNA is postnatally expressed in some tumors, in particular astrocytoma and ganglioneuroblastoma, which are derived from neural tissues not known to express H19 (Ariel et al. supra). Given that H19 RNA is expressed in many types of tumors and cancers, Ariel et al. speculated that H19 RNA was an oncofetal RNA, and proposed investigating H19 as a tumor marker for human neoplasia.
H19 is significantly expressed in 84% of human bladder carcinomas, expression decreasing with tumor loss differentiation. Independent of tumor grade, the H19 expression level significantly correlated with early tumor recurrence (Ayesh, B., et al, Mol Ther, 2003. 7(4): p. 535-41).
Comparing patterns of gene expression in two homogeneous cell populations that differ only in the presence or absence of H19 RNA have identified a plethora of downstream effectors of H19 RNA. Among these are group of genes that were previously reported to play crucial roles in some aspects of the tumorigenic process. H19 RNA presence may enhance the invasive, migratory and angiogenic capacity of the cell by up-regulating genes that function in those pathways, and thus could contribute at least to the initial steps of the metastatic cascade. Additional studies highlight the potential role of H19 in promoting cancer progression and tumor metastasis by being a gene responsive to Hepatocyte growth factor/scatter factor (HGF/SF).
Specific expression of the H19 gene in cancer cells has prompted its use in clinical applications for diagnosing cancer. For example, U.S. Pat. No. 5,955,273 teaches the use of H19 gene as a tumor specific marker. PCT Pub. No. WO 2004/024957 discloses the use of H19 for the detection, in a patient suspected of having cancer, of the presence of residual cancer cells or micro-metastases originating from solid tumors.
IGF-II
Insulin-like growth factor-II (IGF-II) is expressed in the majority of bladder carcinomas such as transitional cell carcinomas (TCC; Ariel, I., et al., The imprinted H19 gene is a marker of early recurrence in human bladder carcinoma. Mol Pathol, 2000. 53(6): p. 320-3). The biological activities are mediated by the binding to the cell surface-receptors IGF-I receptor (IGF-1R), IGF-II receptor (IGF-2R) and insulin receptor (IR). The IGF receptors are present almost in all tissues of fetal and adult animals. IGF-2R binds IGF-II with the highest affinity, whereas the IGF-1R and IR possess high, but lower affinity to IGF-II than to their respective ligands. IGF-II is a potent embryonic and tumor growth factor that signals via the IGF1R through the Ras/mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt/FOXO, and S6K/mammalian target of rapamycin (mTOR) signaling pathways to modify cell proliferation, cell survival, gene expression, and cell growth.
IGF-II is another imprinted gene whose expression depends upon its parental origin. However in contrast to H19, IGF-II is maternally imprinted in both mice and humans, and is therefore expressed from the paternally inherited allele (Rainier et al., 1993, “Relaxation of imprinted genes in human cancer”, Nature 362:747 749). The human IGF-II gene exhibits a complex transcriptional pattern. There are four IGF-II promoters that are activated in a tissue-specific and developmentally specific manner. Only three of the IGF-II promoters (i.e., P2, P3 and P4) are imprinted and active during fetal development and in cancer tissues. The P3 promoter of the IGF-II gene has been implicated in the progression of liver cirrhosis and hepatocellular carcinoma (Seo et al., 1998, “Different protein-binding patterns in the P3 promoter region of the human insulin-like growth factor II gene in the human liver cirrhosis and hepatocellular carcinoma tissues”, J Korean Med Sci.13:171 178).
The fourth IGF-II promoter, (i.e., P1) is not imprinted, and is activated in the adult liver and choroid plexus (See Holthuizen et al., 1993, “Transcriptional regulation of the major promoters of the human IGF-II gene”, Mol Reprod Dev. 35:391 393).
Loss of imprinting of IGF-II has been implicated in Wilm's tumor (Ogawa et al., 1993, “Relaxation of insulin-like growth factor II gene imprinting implicated in Wilm's tumour”, Nature 362:749 751). This observation led many investigators to speculate that the loss of imprinting and biallelic expression of imprinted genes may be involved in growth disorders and the development of cancer (Rainier et al., 1993, Nature 362:747 749; Glassman et al., 1996, “Relaxation of imprinting in carcinogenesis”, Cancer Genet Cytogenet. 89:69 73).
Epigenetic modification and mutations of the IGF-II signaling system occur in cancers such as human colorectal tumors (Hassan A B, Macaulay V M. The insulin-like growth factor system as a therapeutic target in colorectal cancer. Ann Oncol 2002; 13:349-56). Supply of IGF-II is frequently up-regulated, and serial analysis of gene expression has shown IGF-II as a commonly overexpressed gene in a number of cancer cell lines and tumors, e.g. human bladder carcinoma and colorectal cancer (Zhang L, Zhou W, Velculescu V E, et al. Gene expression profiles in normal and cancer cells. Science 1997; 276:1268-72).
WO 99/18195 and U.S. Pat. No. 7,041,654 teach the specific expression of heterologous sequences, particularly genes encoding cytotoxic products (e.g. Diphtheria toxin), in tumor cells under the control of a cancer specific promoter (e.g., an H19 promoter and enhancer, IGF-II P3 promoter, IGF-II P4 promoter, or IGF-1 promoter).
WO 04/031359 teaches a method for regulating the expression of angiogenesis-controlling genes in cells that are involved in neo-vascularization, comprising administering to the cells an effective amount of an H19 modulator.
WO 2007/034487 discloses a nucleic acid construct comprising: (i) a first nucleic acid sequence encoding TNF alpha; (ii) a second nucleic acid sequence encoding a Diphtheria toxin; and (iii) at least one additional nucleic acid sequence comprising a cancer specific promoter (e.g. H19, IGF-1, P3, or IGF-II P4 promoters); the TNF alpha and Diphtheria toxin encoding sequences being under an expression control of the cancer specific promoter. Also provided are construct systems and methods and uses of same.
WO 2007/007317 discloses isolated oligonucleotides capable of down-regulating a level of H19 mRNA in cancer cells, articles of manufacture comprising agents capable of downregulating H19 mRNA in combination with an additional anti-cancer treatment as well as methods of treating cancer by administering same. WO 2007/007317 discloses that anti-cancer drugs can be co-administered with the claimed oligonucleotides.
WO 2008/087641 discloses compositions and methods for treating rheumatoid arthritis, utilizing H19-silencing nucleic acid agents such as inhibitory RNA.
WO 2008/087642 discloses compositions and methods for the treatment of cancer and other conditions that are associated with elevated expression of the H19 gene, utilizing H19-silencing nucleic acid agents such as inhibitory RNA.
WO 2008/099396 discloses compositions and methods for treating restenosis, utilizing H19-silencing nucleic acid agents such as inhibitory RNA.
None of the above references discloses or suggests a single construct containing multiple Diphtheria toxin-expressing open reading frames, wherein the Diphtheria toxin is expressed from a plurality of promoters.
Use of a single promoter (e.g. an H19 promoter or an IGF-II P3 or P4 promoter) alone for expression of a cytotoxic or cytostatic gene from an anti-cancer therapeutic construct presents several unresolved problems. For one, not every tumor of a given type of cancer (e.g. bladder carcinoma, superficial bladder cancer, etc.) is positive for expression via the H19 promoter or the IGF-II P3 or P4 promoter. Thus, such therapy is bound to fail in a sizable proportion of patients, even without accounting for tumor mutagenesis. Determination of responsiveness to such constructs would involve the costly and difficult step of genotyping individual tumors.
Tumors are known to exhibit significant genomic instability and heterogeneity. Thus, even individuals with an H19-expressing tumor, for example, are likely to contain a sizable number of cancer cells that have downregulated or abrogated H19 expression via mutation. Therefore, expressing the cytotoxic or cytostatic gene from a single promoter in such patients may result in temporary and partial tumor regression that will rapidly be reversed when the cells containing these mutations survive and rapidly multiply.
There remains an unmet medical need for developing additional safe and effective therapeutic modalities useful in cancer therapy.
The inclusion or description of literary references in this section or any other part of this application does not constitute an admission that the references are regarded as prior art to this invention.